An analogue of the proposed method [U.S. Pat. No. 4,558,012, Int. Cl. G01N 33/54, U.S. Cl. 436/501, 1985] is known that is intended for detection of chemical material components and measuring their concentration by detection of their binding to a sensor layer, which comprises:                irradiating of the sensor layer by light of various wavelengths, for which the sensor layer is transparent, at least, partially;        registration in the reflected light of a signal, which depends upon optical thickness of the said sensor layer and is due to the fact that interference on the said sensor layer modulates the reflection spectrum of the said sensor layer;        judging about the binding being detected from a change of the said signal.        
In this method, the sensor layer is formed on a non-metallic substrate with high optical absorption, made preferably of a semiconductor, dark glass or plastic. The sensor layer consists of a number of transparent dielectric layers, a material that binds the chemical substance being detected, and the said chemical substance itself that forms a thin near-surface layer as a result of the said binding. The thickness of the sensor layer is chosen so that it acts as an antireflecting coating for polychromatic light of wavelengths within the range of (525-600) nm, which is incident to the said sensor layer. Interference on the sensor layer results in a reflection minimum in the said range. A change of the thickness of the sensor layer due to binding of the said chemical substance results in a spectral shift of the said minimum and, consequently, a change of color of the reflected light. This color change is registered visually and used for judging about presence or concentration of the chemical substance being detected.
Drawbacks of the analogue and the apparatus for its embodiment [U.S. Pat. No. 4,558,012, Int. Cl. G01N 33/54, U.S. Cl. 436/501, 1985] are its low sensitivity, not sufficient reliability and low precision of the results. This is due to qualitative, not quantitative, evaluation of the result and subjective character of the visual evaluation of the color change. Besides, these method and apparatus do not permit real-time registration of binding of chemical substances and investigation of kinetics of the process.
Another analogue [U.S. Pat. No. 4,820,649, Int. Cl. G01N 33/53, U.S. Cl. 436/501, 1989] of the proposed method is known that is intended for detecting components of biological systems, which comprises:                irradiating the sensor layer by light of various wavelengths, for which the sensor layer is transparent, at least, partially;        registration in the reflected light of a signal, which depends upon optical thickness of the said sensor layer and is due to the fact that interference on the said sensor layer modulates the reflection spectrum of the said sensor layer;        judging about the binding being detected from a change of the said signal.        
The method of the analogue [U.S. Pat. No. 4,820,649, Int. Cl. G01N 33/53, U.S. Cl. 436/501, 1989] slightly differs from the method of the analogue [U.S. Pat. No. 4,558,012, Int. Cl. G01N 33/54, U.S. Cl. 436/501, 1985] in that it is applicable to substrates with high reflectivity, in particular, to metallic substrates. Matching the intensities of the light reflected from two boundary surfaces of the sensor layer, which is necessary for effective interference on the sensor layer and producing a clearly distinctive color, is achieved by employment of a semitransparent reflective film of small metallic particles. This metallic film is deposited onto the sensor layer after binding of the component being detected. This method and the apparatus for its embodiment [U.S. Pat. No. 4,820,649, Int. Cl. G01N 33/53, U.S. Cl. 436/501, 1989] have the same drawbacks as the method and apparatus [U.S. Pat. No. 4,558,012, Int. Cl. G01N 33/54, U.S. Cl. 436/501, 1985]. Moreover, they are also more complex and less reliable because of using of the said metallic film.
The closest to the proposed method is a method of optical detection of binding of at least one material component to a substance located on a surface of or inside the sensor layer on the basis of a biological, chemical or physical interaction [DE 42 00 088 C2, Int. Cl. G01N 21/45, 1997], which comprises:                irradiating the sensor layer by light of various wavelengths, for which the sensor layer is transparent, at least, partially;        registration in the reflected or transmitted light of a signal, which depends upon optical thickness of the said sensor layer and is due to the fact that interference on the said sensor layer modulates the reflection or transmission spectrum of the said sensor layer, respectively;        recording the spectrum of the said reflected or transmitted light as the said signal;        judging about the binding being detected from a change of the said signal.        
According to this method, the sensor layer is located on a sufficiently transparent substrate and is irradiated by light of appropriate wavelengths from the side of the substrate. The sensor layer consists of, at least, partially, a layer of transparent inorganic (e.g. oxides, nitrides) or organic polymer (e.g. polystyrene) and a substance that implements the binding to be detected. The said substance is located on the surface of or inside the sensor layer and is capable to bind the said material component. Reaction of specific binding of an antibody with an antigen can be mentioned as an example of such binding. A material that enhances reflection is placed between the sensor layer and the substrate. The material forms one boundary surface of the sensor layer. The other surface is formed by an external medium. The external medium is commonly a biological solution under test, which contains or presumably contains the said component, whose binding is the object of detection.
Interference on the sensor layer results from combining of two or more secondary light waves produced as a result of partial reflection and partial transmission on the boundary surfaces of the sensor layer and, probably, on the interface surfaces inside the sensor layer. Said interference modulates the reflection and transmission spectra of the sensor layer. The spectrum of the reflected or transmitted light is recorded, and the absolute optical thickness of the sensor layer is determined from shape of the spectrum by analytical fitting. Information about a change of optical thickness of the sensor layer due to the binding being detected and, consequently, about parameters of the said binding is obtained from a change of the recorded spectrum.
Unlike analogues [U.S. Pat. No. 4,558,012, Int. Cl. G01N 33/54, U.S. Cl. 436/501, 1985] and [U.S. Pat. No. 4,820,649, Int. Cl. G01N 33/53, U.S. Cl. 436/501, 1989], the method-analogue [DE 42 00 088 C2, Int. Cl. G01N 21/45, 1997] provides capability of real-time detection of binding of material components to a substance of the sensor layer and detachment of the said components from the substance of the sensor layer, which is an important advantage of the method-analogue.
In the method-analogue, the sensor layer must be thin, i.e. there are a number of limitations implied on its thickness:                the thickness is of the same order of magnitude as the wavelength of the used light;        the double thickness is less than the coherence length of the used light;        the thickness is within the range (0.3-10) μm, being not more than 5 μm in important practical cases and 2 μm in preferable variants.        
The mentioned limitations are due to the principle of how the absolute thickness of the sensor layer is determined in the method-analogue. Employment of thicker sensor layers would result in interference pattern with many periods in the recorded spectrum. Unambiguous determination of the absolute thickness of the sensor layer from such interference pattern would be difficult or impossible.
The mentioned principle and related limitations on thickness of the sensor layer give rise to a number of drawbacks of the method-analogue. The spectral dependence of intensity of the reflected or transmitted light that serves for determination of the absolute optical thickness in the method-analogue represents a smooth curve that slowly varies within the observed spectral range. Due to this fact, any intensity variations in the recorded spectrum lead to significant errors in the measurement results. This particularly refers to variations that are non-uniform over spectrum. They can arise from drifts of operating parameters of the radiation source, changes of its temperature, heating of optical elements of the scheme, thermal and mechanical instabilities of the optical scheme due to changes of ambient conditions, etc.
Application of the method-analogue to multi-channel registration of structural changes of substances of the sensor layer including binding of material components to one or several substances of the sensor layer is known [U.S. Pat. No. 5,999,262, Int. Cl. G01B 9/02, U.S. Cl. 356/357, 1999]. In this method, the said structural changes in several spatially separated areas of the sensor layer are detected, and all the said areas are simultaneously irradiated by the light of the said wavelengths. The spectrum of the said reflected or transmitted light is recorded for each said area by using sequentially in time different wavelengths and implementing the following operations for each of the said wavelengths: irradiating the said areas by monochromatic light of one wavelength and measuring intensity of the said reflected or transmitted light for each said area.
As this takes place, the sensor layer is placed either on a plate-substrate, which is positioned on a base plate while measuring, or directly on the base plate. This is due to the fact that at the multi-channel registration [U.S. Pat. No. 5,999,262, Int. Cl. G01B 9/02, U.S. Cl. 356/357, 1999] the sensor layer is also thin, and the same limitations as in the method-analogue [DE 42 00 088 C2, Int. Cl. G01N 21/45, 1997] are implied on its thickness.
The method of multi-channel registration is based on analysis of form of the recorded spectrum in each channel and, accordingly, determining of the absolute optical thickness of the sensor layer in each area. This means that the method-analogue [DE 42 00 088 C2, Int. Cl. G01N 21/45, 1997] is used for every channel (each area of the sensor layer under study). In this case, all the mentioned drawbacks of the method-analogue remain in its multi-channel variant. Moreover, they manifest themselves even stronger. Since in the method of multi-channel registration different spectral regions are recorded sequentially in time, any drifts and instabilities in intensity of the analyzed light in whole or in any region of its spectrum result in lower accuracy of measurements. Besides, as the multi-channel registration requires a powerful light source, the negative role of thermal instabilities of all elements of the optical scheme, namely: the source, detector, dispersion elements or spectral filters, assembly elements, etc. sharply increases. Such instabilities cause not only drifts of spectral distribution of the light intensity in each channel, but also drifts of intensity distribution of the analyzed light over the channels. Among characteristic examples, one can mention a change of color temperature of the light source due to heating of a filament and a change of intensity distribution of exposure along surface of the sensor layer and along surface of the photodetector, where images of different areas of the sensor layer are transformed, due to the filament sag because of heating. In these cases, uncontrollable drifts of both spectrum of the analyzed light and intensity distribution over the registration channels take place.
All this leads to low sensitivity, insufficient resolution, low reliability and precision of results obtained by the method-analogue [DE 42 00 088 C2, Int. Cl. G01N 21/45, 1997] and especially its multi-channel variant [U.S. Pat. No. 5,999,262, Int. Cl. G01B 9/02, U.S. Cl. 356/357, 1999]. Complexity, high labor input and cost can be mentioned among drawbacks of the method and, even more, its multi-channel variant.
Variants of the apparatus that realizes the proposed variants of the method are proposed.
The closest to the proposed apparatus is an apparatus-analogue intended for optical detection of binding of at least one material component to a substance located on a surface of or inside the sensor layer on the basis of a biological, chemical or physical interaction [DE 42 00 088 C2, Int. Cl. G01N 21/45, 1997], which comprises:                a sensor layer;        a source of light, which irradiates the sensor layer, of wavelengths that include at least operating wavelengths, for which the sensor layer is transparent, at least, partially;        a detector, which is placed on the pathway of the light reflected from the sensor layer or transmitted through the sensor layer, for measuring the light intensity of operational wavelengths in the spectrum of the received light;                    a block of result generation, for example, a computer, to generate information about the binding being detected on the basis of changes of the said spectrum, whose input is connected to the output of the detector.                        
In the apparatus-analogue, the sensor layer is located on a transparent substrate, which is made preferably of glass, and the light from the source irradiates the sensor layer from the substrate's side. The sensor layer comprises a transparent, preferably inorganic, optical substance and a substance that binds a detected material component on the surface of or inside the sensor layer. A spectrometer preferably made on the basis of a photodiode array is used as a detector to register reflection or transmission spectrum of the sensor layer. The block of the result generation in the apparatus-prototype is made capable to determine the absolute thickness of the sensor layer from the recorded spectrum that is modulated by interference on the sensor layer.
As was discussed above during consideration of the method-analogue [DE 42 00 088 C2, Int. Cl. G01N 21/45, 1997], operation of the apparatus-analogue is based on the fact that binding being detected of material components on the surface of or inside the sensor layer changes optical thickness of the sensor layer. The block of result generation in specified moments of time determines the absolute optical thickness of the sensor layer from the spectrum recorded by a detector, and informs about the binding being detected based on a change of this absolute thickness.
The used in the apparatus-analogue principle of determining of absolute thickness imposes a number of simultaneous restrictions on thickness of the sensor layer:                thickness is of order of magnitude of the wavelength of the used light;        double thickness is less than the coherence length of the used light;        thickness is in the range (0.3-10) in practically important cases and preferable variants not exceeding 5 μm and 2 μm, respectively.        
The mentioned peculiarities and restrictions of the apparatus-analogue give rise to a number of drawbacks discussed earlier while analyzing the corresponding method, namely, significant errors in measurement results due to uncontrollable intensity variations over the recorded spectrum or its intervals, low sensitivity and resolution, low precision and insufficient reliability of results of measurements.
A multi-channel variant of the apparatus-analogue is also known [U.S. Pat. No. 5,999,262, Int. Cl. G01B 9/02, U.S. Cl. 356/357, 1999], which is intended for investigation of structural changes, in particular, binding of material components, in several areas of the sensor layer. In the said apparatus, a source is monochromatic and tunable; the sensor layer is arranged either on a carrier plate or on a substrate placed on the carrier plate for measurements; the light irradiates simultaneously all the areas under study; a detector represents a set of photoelectric detectors; a control link is introduced between the block of result generation and the source to switch the latter to another wavelength of irradiated light after measurement by the detector of the intensity of received light for each said area at one wavelength; the block of the result generation is made capable to generate a spectral distribution of intensity of the received light over wavelength for each said area; judgment about binding being detected is made based on a change of the said spectral distribution for each said area.
Principle of operation of this apparatus consists in point-by-point determination of the spectrum for each area under study. At first, intensity in the spectrum is measured for all areas at one wavelength, then at another wavelength, etc. One determines absolute thickness of the sensor layer from the obtained spectra for each area and judges about binding being detected from a change of this thickness for each area.
A microtiter plate is also known [U.S. Pat. No. 6,018,388, Int. Cl. G01N 21/03, U.S. Cl. 356/246, 2000] for multi-channel registration of binding processes, which is a base component of the apparatus [U.S. Pat. No. 5,999,262, Int. Cl. G01B 9/02, U.S. Cl. 356/357, 1999]. In this plate, a sensor layer is up to 1 μm thick and formed on a bottom plate. There is at least one more layer between the sensor layer and the bottom plate. Back side of the bottom plate has an anti-reflecting coating. Areas of the sensor layer, for which the said binding is studied, are formed by a non-detachable joint of the bottom plate and a second plate that has a number of holes. The sensor layer in each area forms the bottom of a reaction cell while the holes of the second plate form side walls of these reaction cells.
Mentioned variants of the apparatus [U.S. Pat. No. 5,999,262, Int. Cl. G01B 9/02, U.S. Cl. 356/357, 1999 and U.S. Pat. No. 6,018,388, Int. Cl. G01N 21/03, U.S. Cl. 356/246, 2000] employ the same principle of determination of thickness of the sensor layer as the apparatus-analogue [DE 42 00 088 C2, Int. Cl. G01N 21/45, 1997] and impose the same restrictions on the thickness of the sensor layer. As it was discussed above during analysis of the method [U.S. Pat. No. 5,999,262, Int. Cl. G01B 9/02, U.S. Cl. 356/357, 1999], all drawbacks of the apparatus-analogue [DE 42 00 088 C2, Int. Cl. G01N 21/45, 1997] are inherent to the apparatus [U.S. Pat. No. 5,999,262, Int. Cl. G01B 9/02, U.S. Cl. 356/357, 1999], including realization [U.S. Pat. No. 6,018,388, Int. Cl. G01N 21/03, U.S. Cl. 356/246, 2000], and they are even more pronounced.
Thus, the required technical result is to make measurement results independent from uncontrollable variations of intensity of the analyzed light in whole as well as in some parts of the spectrum and some regions of the sensor layer surface, and, consequently, to increase accuracy and reliability of the measurements, sensitivity and resolution with simultaneous reduction of a number of necessary operations, decreasing of labor-input and cost of the method and apparatus in both single- and multi-channel variants including real-time operation.